In recent years, nucleic acid aptamers, as with other functional nucleic acids such as siRNAs, have received attention as novel active ingredients for pharmaceutical drugs or diagnostic drugs in place of low-molecular-weight compounds and are under research and development in various ways around the world with the aim of medically applying the aptamers.
These nucleic acid aptamers are functional nucleic acids capable of strongly and specifically binding, through their own conformations, to target substances such as proteins to inhibit or suppress the functions of the target substances. The nucleic acid aptamers are typically produced as nucleic acid molecules binding to target substances from nucleic acid libraries comprising random nucleotide sequences by an in vitro selection method called SELEX (systematic evolution of ligands by exponential enrichment) (Patent Literatures 1 to 3 and Non Patent Literatures 1 to 4).
Conventional nucleic acid aptamers are predominantly RNA aptamers composed of RNAs. The RNAs, however, are unstable and are produced at high cost. For these reasons, research and development have been being shifted in recent years to DNA aptamers, which are stable in vivo and can be inexpensively produced (Patent Literature 4 and Non Patent Literatures 5 to 8). Nevertheless, the DNA aptamers are difficult to produce efficiently, compared with the RNA aptamers. The SELEX method typically involves a method for isolating a complex formed by the binding between the target substance and the nucleic acid aptamer and adopts, as this method, (1) a method which involves trapping proteins onto a nitrocellulose filter through the use of hydrophobic interaction to thereby recover the complex, (2) a method which involves recovering the complex on the basis of mobility shift on a gel during gel electrophoresis, or (3) a method which involves labeling in advance target substances, immobilizing the target substances onto an affinity support or the like on the basis of the labels, and mixing the resulting support with a DNA library.
DNAs, however, are more hydrophobic than RNAs and are therefore nonspecifically adsorbed onto the nitrocellulose filter. In this respect, the method (1) exhibits an undesired high background. The method (2) is unsuitable for the formation of target substance-DNA complexes in large volumes, because possible electrophoresis is limited by gel size. In addition, the DNA library, which consists of plural types of different sequences, tends to produce disturbed electrophoretic bands. Unlike the nitrocellulose filter method, disadvantageously, this method does not permit washing operation of the trapped complex. The method (3) fails to yield aptamers having high binding ability, because DNAs hardly bind to the solid phase-bound surface of the target substances. In addition, DNAs bound both with the target substance and with the solid-phase support are obtained, resulting in undesired high background.